大鼠泰泽氏菌套式PCR方法的建立和初步应用

Establishment and Preliminary Application of Nested Polymerase Chain Reaction for Bacillus piliformis

根据泰泽氏菌 r DNA序列设计出二对特异引物 ,以标准菌 RJ株和大肠杆菌、溶血性链球菌、嗜肺巴氏杆菌纯化的 DNA为模板 ,进行套式 PCR反应。结果用外引物扩增 ,泰泽氏菌和大肠杆菌均出现 62 5 bp的反应带 ,其它细菌无反应带 ;而用内引物第二次扩增 ,只有泰泽氏菌出现 1 96bp的特异扩增带。将 PCR方法应用于人工免疫抑制的 SD大鼠 ,检出率为 3 0 % ( 9/ 3 0 ) ,同时将此 3 0份血清用间接免疫荧光法 ( IFA)进行检测 ,结果未检出阳性样品。结果提示 ,套式 PCR方法具有特异性强、敏感性高 ,简便快速的特点。

On the basis of the 16S ribosomal DNA(rDNA) sequences, the DNA extracted from Bacillus piliformis, Escherichia coli., Hemolytic streptococcus, Pasteuralla pneumotroopica were amplified by PCR using 2 pairs of PCR specific primers. With outer-primers, a DNA fragment of the expected size (625bp) was amplified from Bacillus piliformis and E. coli, but no band was observed in Hemolytic streptococcus and Pasteuralla pneumotroopica. Whereas a 196bp fragment was obtained only from Bacillus piliformis with inner-primers. Thirty liver samples from immunosuppressive rats were detected for Bacillus piliformis by using nested PCR, the positive rate is 30%(9/30), but no serum sample is positive monitored by IFA.In conclusion, the 16S rDNA-based PCR established is sensitive and specific in detcction of Bacillus piliformis in infected animals.